Project Abstract Exposure to unpredictable and complex stress early in life increases the risk for developing anxiety disorder and abnormal connectivity between the amygdala, the prefrontal cortex (PFC), and the hippocampus (HPC). Exactly how early life stress (ELS) modifies fronto-limbic connectivity and how these changes contribute to increased anxiety are questions that are difficult to investigate in humans. Specifically, the question of how ELS interacts with sex to alter fronto-limbic connections and anxiety are not addressable with current human studies. To clarify these issues we developed a mouse model of ELS that we have named Unpredictable Postnatal Stress (UPS), in which mice are exposed to unpredictable and complex stress early in life. We found that UPS caused robust increase in anxiety that was not seen in mice exposed to simple and predictable form of ELS, known as limited bedding (LB). Moreover, we found that UPS and LB preferentially increased anxiety in male mice. Using resting state fMRI (rsfMRI), high resolution diffusion MRI (dMRI), and retrograde tracing we found increased connectivity in male UPS mice that was present during the juvenile period and adulthood and was highly correlated with anxiety. Preliminary data with dMRI indicate that fronto-limbic connectivity is not altered in females and that these sex specific effects are associated with abnormal synaptic pruning and reduced levels of the transcription factor IRF8 in male microglial, but not female microglia. We hypothesize that amygdala connectivity with the PFC and the HPC undergoes microglial-mediated pruning during the postnatal period. This process requires high levels of the transcription factor IRF8 in postnatal microglia and is critical for programming levels of anxiety during the juvenile period and adulthood. UPS reduces expression of IRF8 in males, but not female mice. This in turn leads to increase fronto-limbic connectivity and anxiety in UPS males, but not UPS females. Work in aim 1 will use rsfMRI, dMRI, and dual retrograde tracing to characterize the effects of UPS, sex and age on fronto-limbic connectivity. Studies in aim 2 will test whether reducing IRF8 in postnatal microglia is sufficient to phenocopy the effects of UPS on anxiety, fronto-limbic connectivity, microglial gene expression, and phagocytic activity. In aim 3 we ask whether overexpressing IRF8 in postnatal microglia can normalize anxiety and fronto-limbic connectivity in UPS mice. Successful completion of this application will define a novel role for microglial IRF8 in refining fronto-limbic connections and programming anxiety in the mouse; an important finding given the conserved role that IRF8 plays in guiding immune responses in humans and mice. In addition, this work will provide a new paradigm to explain how ELS differentially affects microglial function, amygdala connectivity, and anxiety in mice.